New use of specific cyklolignans

ABSTRACT

The invention refers to the use of specific cyclolignans, wherein the carbon atoms in positions 9 and 9′ have cis configuration, for inhibition of the insulin-like growth factor-1 receptor. Said compounds can be used for treatment of IGF-1R dependent diseases, such as cancer, psoriasis, artherosclerosis and acromegaly. A preferred compound is picropodophyllin.

CONTINUATION DATA

This application is a Continuation of U.S. application Ser. No.12/508,218, filed on Jul. 23, 2009, which is a Divisional of U.S.application Ser. No. 12/021,530, filed on filed Jan. 29, 2008, which isa Continuation of U.S. application Ser. No. 10/481,441, filed on Dec.19, 2003, which is a National Stage of PCT/SE02/01202, filed on Jun. 19,2002, which claims benefit to U.S. Provisional Application Ser. No.60/300,431, filed on Jun. 26, 2001, each of which is incorporated hereinby reference.

The present invention refers to the use of specific cyclolignansinhibiting the insulin-like growth factor-1 receptor, the IGF-1R, fortreatment of IGF-1R dependent diseases, especially cancer.

BACKGROUND OF THE INVENTION

The insulin-like growth factor-1 receptor (IGF-1R) plays an importantrole in proliferation, protection against apoptosis and transformationof malignant cells. The IGF-1R is also important for maintaining themalignant phenotype of tumour cells, and is involved in tumour cellprotection against anti-tumour therapy. In contrast, the IGF-1R does notseem to be an absolute requirement for normal cell growth.

The IGF-1R consists of two identical extracellular a-subunits that areresponsible for ligand binding, and two identical β-subunits withtransmembrane domain and an intracellular tyrosine kinase domain. Theligand-receptor interaction results in phosphorylation of tyrosineresidues in the tyrosine kinase domain, which spans from amino acid 973to 1229, of the β-subunit. The major sites for phosphorylation are theclustered tyrosines at position 1131, 1135 and 1136 (LeRoith, D., etal., Endocr Rev 1995 April; 16 (2), 143-63). After autophosphorylationthe receptor kinase phosphorylates intracellular proteins, like insulinreceptor substrate-1 and Shc, which activate the phosphatidyl inositol-3kinase and the mitogen-activated protein kinase signalling pathways,respectively. Based on the pivotal role of IGF-1R in malignant cells, itbecomes more and more evident that IGF-1R is a target for cancer therapy(Baserga, R., et al., Endocrine Vol. 7, no. 1, 99-102, August 1997). Onestrategy to interfere with IGF-1R activity is to induce selectiveinhibition of the IGF-1R tyrosine kinase. However, there are today noavailable selective inhibitors of IGF-1R.

Drugs containing the notoriously toxic cyclolignan podophyllotoxin havebeen used for centuries, and its anti-cancer properties have attractedparticular interest. Undesired side effects of podophyllotoxin have,however, prevented its use as an anti-cancer drug. The mechanism for thecytotoxicity of podophyllotoxin has been attributed to its binding toβ-tubulin, leading to inhibition of microtubule assembly and mitoticarrest. The trans conformation in the lactone ring of podophyllotoxinhas been shown to be required for binding to β-tubulin. In contrast, itsstereoisomer picropodophyllotoxin, which has a cis configuration in thelactone ring, has a 50-fold lower inhibitory effect on microtubulepolymerisation and a more than 35-fold higher LD50 in rats. Because ofthe low anti-microtubule effect of picropodophyllotoxin this compoundhas attracted little interest. During the last decades the majorinterest on podophyllotoxin derivatives has concerned etoposide, whichis an ethylidene glucoside derivative of4′-demethyl-epi-podophyllotoxin. Etoposide, which has no effect onmicrotubules, is a topoisomerase II inhibitor, and is currently beingused as such in cancer therapy.

PRIOR ART

A number of synthetic tyrosine kinase inhibitors, that is tyrphostins,have been studied by Párrizas, M., et al., Endocrinology 1997, Vol. 138,No. 4, 1427-1433. The IGF-1R is a member of the tyrosine kinase receptorfamily, which also includes the receptors of insulin, epidermal growthfactor (EGF), nerve growth factor (NGF), and platelet-derived growthfactor (PDGF). All of the tyrphostins active on IGF-1R cross-react withthe insulin receptor, although two of them showed a moderate preferencefor IGF-1R. It was therefore suggested that it could be possible todesign and synthesize small molecules capable of discriminating betweenthem.

Substrate competitive inhibitors of IGF-1 receptor kinase are discussedby Blum, G., et al. in Biochemistry 2000, 39, 15705-15712. A number oflead compounds for inhibitors of the isolated IGF-1R kinase arereported. The search for these compounds was aided by the knowledge ofthe three-dimensional structure of the insulin receptor kinase domain,which is 84% homologous to the IGF-1R kinase domain. The most potentinhibitor found was tyrphostin AG 538, with an IC50 of 400 nM. However,said inhibitor also blocked the insulin receptor kinase.

Kanter-Lewensohn, L., et al., Mol Cell Endocr 165 (2000), 131-137,investigated whether the cytotoxic effect of tamoxifen (TAM) on melanomacells could depend on interference with the expression or function ofthe insulin-like growth factor-1 receptor. It was found that, althoughTAM did not have a strong effect on IGF-1 binding and the expression ofIGF-1R at the cell surface, TAM efficiently blocked tyrosinephosphorylation of the IGF-1R β-subunit.

The Chemistry of Podophyllum by J. L. Hartwell et al., Fortschritte derChemie organischer Naturstoffe 15,1958,83-166, gives an overview ofpodophyllotoxin and different derivatives thereof, commercially derivedfrom two species of plants, Podophyllum peltatum and Podophyllum emodi.

Picropodophyllin has generally been considered to be biologicallyinactive, Ayres, D. C., and Loike, J. D., Lignana. Chemical, biologicaland clinical properties. Cambridge University Press, Cambridge, 1990.

OBJECTS OF THE INVENTION

The object of the invention is to find new methods for treatment ofIGF-1R dependent diseases, especially cancer, by means of an inhibitionof the tyrosine kinase of the insulin-like growth factor-1 receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a computer model of a 12 amino acid peptide comprising thetyrosines 1131, 1135 an 1136 of the IGF-1 receptor.

FIG. 2A shows the structural formulas of the compounds picropodophyllinand podophyllotoxin, and FIG. 2B the structural formulas ofdeoxypicropodophyllin and β-apopicropodophyllin.

FIG. 3 is a diagram showing the effect of picropodophyllin on tyrosinephosphorylation of different receptors.

FIG. 4 is a diagram showing the effect of picropodophyllin on theautophosphorylation of IGF-1R.

FIGS. 5A and 5B are diagrams showing the dose-response effects ofpicropodophyllin on the viability of 4 different cell lines. FIG. 5Ashows the effect on a melanoma cell line, FM 55, and a sarcoma cellline, RD-ES, respectively, and FIG. 5B the effect on two manipulatedcell lines, R— and P6.

FIGS. 6A and 6B are diagrams showing the effect of picropodophyllin ontumor weight in mice.

DESCRIPTION OF THE INVENTION

The three-dimensional structure of a 12-amino acid sequence of theIGF-1R tyrosine domain, including the tyrosine residues at position1131, 1135 an 1136, was analysed using a computer programme in order tofind compounds having the ability to mimic the tyrosine residues andinterfere with the phosphorylation thereof. It was then discovered, whenusing a 12-amino acid peptide, that two of the three key tyrosines, thatis 1135 and 1136, which have to be autophosphorylated in IGF-1R foractivation, could be situated as close as 0.95 nm (9.5 Å from eachother, and that the apparent angle between these groups was about 60°.The configuration of said sequence is shown in FIG. 1. Such a shortdistance has not been observed for the corresponding tyrosines in theinsulin receptor. FIG. 1 also depicts the space structure ofpodophyllotoxin and picropodophyllin.

Molecular modelling showed that an inhibitory molecule could consist oftwo benzene rings separated by only one carbon atom. When a two-carbonbridge was tried, the distance between the substituents of the benzenerings was too long, about 1.3 nm (13 Å).

The substituents corresponding to the hydroxy groups in the tyrosineswere selected to be methoxy or methylenedioxy groups, since they arechemically relatively stable, i.e. they are not oxidized orphosphorylated. The distance between these substituents should be about0.95±0.10 nm (9.5±1.0 Å).

It was then surprisingly found that two angled and substituted benzenerings of the cyclolignans podophyllotoxin and picropodophyllin fittedalmost exactly into the pocket between tyrosine 1135 and 1136,indicating that said compounds could interfere with theautophosphorylation of the tyrosine residues. In contrast to the effecton microtubuli, the IGF-1R inhibition was not limited to cyclolignanswith a trans configuration in the lactone ring.

In order to penetrate the receptor, an inhibitory molecule has to besmall. When for instance podophyllotoxin was conjugated with a glucosidederivative, podophyllotoxin-4,6-O-benzylidene-β-D-glucopyranoside, theeffect on IGF-1R completely disappeared. Furthermore, followingreduction of the lactone ring to a diol structure, the size of themolecule increased due to the reduced substituents sticking out from themolecule, resulting in a dramatically reduced activity of the compounds.Increasing the size by forming methylenedioxy derivatives or acetonidesof podophyllotoxin-diol also resulted in compounds with little or noactivity.

The inhibitor molecule also has to be relatively non-polar, so that itcan freely penetrate cell membranes and the IGF-1 receptor, butsufficiently polar to be reasonably soluble in water. The polarity ofthe molecule is determined by the number and nature of the oxygenfunctions. The polarity seems to be optimal when the water solubility isbetween that of deoxypodophyllotoxin, i.e. about 0.01 mM, and that ofpodophyllotoxin, about 0.1-0.2 mM. No charged or highly polar groupsshould be present on the molecule.

The invention refers to a compound of the formula I

wherein R₁, which can be the same or different, is OH or OCH₃, n is 0, 1or 2, R₂, R₃ and R₄, which can be the same or different, are H, OH, O,OOCH₃, OOCH₂CH₃, OCH₃, or OC₂H₅, or R₃ and R₄ together are an ether or alactone, and which optionally contains a double bond Δ⁷⁽⁸⁾ or Δ^(8(8′)),for use as a medicament.

Notably, the carbons in positions 9 and 9′ of all the compounds of theformula I have a cis configuration, i.e. the 8-9 and 8′-9′ bonds arelocated in or above the plane of the carbon ring (beta bonds), asindicated by the solid lines in the formula I. A wavy line, as betweenthe carbons 1′ and 7′, indicates that the bond can be either an alpha ora beta bond. An alpha bond, that is below the plane of the carbon ring,is illustrated by a dashed line. The benzene ring should preferably bein α-position, as is demonstrated by picropodophyllin,deoxypicropodophyllin, α- and β-apopicropodophyllin.

The invention especially refers to compounds of the formula II,

wherein R₂ is defined as in formula I, for use as a medicament.Preferred compounds are picropodophyllin or deoxypicropodophyllin. Thechemical structure of said compounds is shown in FIG. 2A.

Podophyllotoxin and deoxypodophyllotoxin, used as starting material forthe synthesis of the described picro derivatives, are naturallyoccurring in plants. For the preparation of said substances in pureform, dried and finely ground rhizomes of e.g. Podophyllum emodi orPodophyllum peltatum are extracted with organic solvents. The extract isthen filtered and concentrated on silica gel. The fractions containingthe substances are collected and the latter are further purified bychromatography on acid alumina and silica gel etc., and finallyrecrystallized.

Deoxypicropodophyllin and picropodophyllin can be prepared fromdeoxypodophyllotoxin and podophyllotoxin, respectively. One mg of thelatter was dissolved in 70% aqueous methanol. To the solution was added20 mg of sodium acetate and the mixture was then incubated for 20 h at55° C. After evaporation of the alcohol, the product was extracted withethyl acetate, and then purified by chromatography on silica gel, mobilephase: hexane-ethyl acetate mixtures, and/or octadecylsilane-bondedsilica, mobile phase: aqueous methanol; HPLC).

Other compounds of the invention of special interest for use as amedicament can be described by the following formula III

wherein R₃ and R₄ are defined as in formula I, and which in additioncontains a double bond Δ⁷⁽⁸⁾ or Δ⁸⁽⁸⁾, for use as a medicament.Preferred compounds of the formula III are α-apopicropodophyllin andβ-apopicropodophyllin.

α- and β-apopicropodophyllin can be prepared from podophyllotoxin byincubation in a buffered ethanolic solution at elevated temperature, asdescribed by Buchardt, O. et al., J Pharmaceut Sci 75,1076-1080, 1986.The total synthesis of picropodophyllin and its apo derivatives havebeen described by Gensler, J. W., et al., J Am Chem Soc 82, 1714-1727,1960.

As additional examples of compounds of the formula I can be mentioned:epipicropodophyllin, picropodophyllone, 4′-demethylpicropodophyllin, andthe acetate derivative of picropodophyllin and the methyl ester andethyl ester derivatives of picropodophyllic acid.

The invention especially refers to the use of a compound of the formulaI for the preparation of a medicament inhibiting tyrosinephosphorylation of the insuline-like growth factor-1 receptor.

To design an inhibitor of the IGF-1R tyrosine kinase for therapeuticpurposes it is of critical importance that the inhibitor does notcross-react with the insulin receptor kinase, which is highly homologousto the IGF-1R. Co-inhibition of the insulin receptor will lead to adiabetogenic response in vivo. This response comprises a very seriousside effect, which cannot be overcome by insulin treatment since thereceptor kinase is being blocked. It has, however, now beendemonstrated, see FIG. 3, that picropodophyllin, which is a much morepotent IGF-1R inhibitor than the tyrophostin-based compounds, does notat all interfere with the insulin receptor tyrosine kinase. Nor does itinterfere with tyrosine phosphorylation of the epidermal growth factor,platelet derived growth factor or fibroblast growth factor receptors.

According to a preferred aspect the invention refers to the use of acompound as described above for the preparation of a medicament forprophylaxis or treatment of IGF-1R dependent diseases, such as cancer,artheriosclerosis, including prevention of restenosis of the coronaryarteries after vascular surgery, psoriasis and acromegaly.

Podophyllotoxin has for long been implicated in cancer therapy, butproduced unacceptable side effects. The anti-cancer effect, as well asthe side effects, was attributed to inhibition of microtubule assemblyand mitotic block. It has now been demonstrated that podophyllotoxin andits non-toxic isomer picropodophyllin, which has generally beenconsidered to be biologically inactive, are very potent and specificinhibitors of tyrosine phosphorylation of the insulin-like growthfactor-1 receptor, which plays a pivotal role as a survival factor incancer cells of utmost importance is that neither picropodophyllin norother picro derivatives having a cis configuration in the lactone ringinhibit the insulin receptor, which is highly homologous to IGF-1R. Nordo they inhibit other major growth factor receptor kinases. The lowgeneral cytotoxicity of picropodophyllin compared with podophyllotoxinsuggests that the former compound is a very selective IGF-1R inhibitor.

The results of the biological experiments suggest that submicromolarconcentrations of picropodophyllin or other picro derivatives can besufficient to cause tumor cell death. However, for optimal treatment itis believed that it is important to keep a relatively constantconcentration of the inhibitors over lengthy periods, to allow them tocontinuously saturate all IGF-1Rs, and in this way eventually kill asmany malignant cells as possible. Therefore, infusion ofpicropodophyllin or derivatives, in connection with monitoring theplasma concentration of the compound, may be the strategy of choiceinstead of repetetive (e.g. daily) administrations, which may lead toreactivations of IGF-1R between the treatments.

Previous attempts to treat humans and animals with podophyllotoxin havedemonstrated that this is a relatively toxic compound, both systemically(LD₅₀ for rats is 14 mg/kg) and locally (tissue damaging). Itscytotoxicity has been linked to its binding to β-tubulin, but thisshould only occur at much higher concentrations than those required forIGF-1R-inhibition when tested in vitro (IC₅₀ of 0.5-1.0 μM versus IC₅₀of 0.001 μM). This toxicity precludes its use as a drug for parenteral,per oral, and topical administration.

The invention especially refers to the use of a compound as describedfor the preparation of a medicament for prophylaxis or treatment ofdifferent types of cancer, such as malignant melanoma; primitiveneuroectodermal tumors, such as Ewing's sarcoma; gliomas, such asglioblastoma and astrocytoma; prostate carcinoma; breast carcinoma;myeloproliferative and lymphoproliferative diseases, such as leukemiasand lymphomas; gastrointestinal tumors, such as gastric cancer, coloniccancer and pancreatic carcinoma; gynecological cancer, such as ovarialcancer and endometric carcinoma.

In case of tumors not completely dependent on IGF-1R, the compounds ofthe invention can be useful to potentiate the effects of otheranti-cancer drugs. The invention therefore also refers to the use of acompound of the formula I in combination with another cytostaticum. Asexamples of cytostatica which can be used together with a cyclolignan ofthe invention can be mentioned vincristin, taxol and etoposide.

According to a special aspect the invention refers to the use ofcompounds of the formula III for the preparation of a medicament fortreatment of leukemia.

In addition to cancer therapy, the cyclolignans may be valuable fortreatment of other diseases, the pathogenesis of which involvesIGF-1/IGF-1R, like artheriosclerosis and psoriasis, see for instanceBayes-Genis, A., et al., Circ Res 86, 125-30 (2000).

According to a special aspect the invention refers to the use ofcompounds of the formula I for the preparation of a medicament fortreatment of psoriasis. Epidermal hyperplasia is a key feature of thecommon skin disorder psoriasis. Stimulation of epidermal keratinocytesby IGF-1 is essential for cell division and increased sensitivity toIGF-1 may therefore occur in psoriasis. In a recent study IGF-1Rantisense oligonucleotides were injected into human psoriasis lesions,and this treatment caused a dramatic normalization of the hyperplasticepidermis, Wraight, C. J., et al.; Nat Biotechnol 18, 521-6 (2000). Thisstrongly suggest that IGF-1R stimulation is a rate-limiting step inpsoriatic epidermal hyperplasia and that IGF-1R targeting by selectiveinhibitors can form the basis of a potential new psoriasis therapy.

According to another aspect the invention refers to the use of compoundsof the formula I for the preparation of a medicament for treatment ofartheriosclerosis and restenosis following coronary angioplasty. IGF-1is a growth promoter for arterial cells and a mediator of cardiovasculardisease, such as artheriosclerotic plaque development and restenosisafter coronary angioplasty. IGF-1 promotes macrophage chemotaxis, excessLDL cholesterol uptake, and release of proinflammatory cytokines.Furthermore, IGF-1 stimulates vascular smooth muscle cell (VSMC)proliferation and migration to form a neointima. Thus, IGF-1 seems toplay a key role in these events and in order to limit or reverse plaquegrowth and vulnerability in artheriosclerosis and in the neointimalhyperplasia of restenosis, the activity of IGF-1 can be suppressed byusing the IGF-1R inhibitors. (Bayes-Genis, A., et al., Circ Res 86,125-130, 2000.)

The invention also refers to a pharmaceutical composition comprising acompound of the formula I in combination with a physiologicallyacceptable carrier. The pharmaceutical composition, which optionallycontains conventional additives, can be administered to a patient by anysuitable route, depending on the disease being treated and the conditionof the patient.

For parenteral administration, the compounds may be administered asinjectable dosages or by continuous intravenous infusion of a solution,suspension or emulsion of the compound in a physiologically acceptablediluent as the pharmaceutical carrier, which can be a sterile liquid,such as water, alcohols, oils, and other acceptable organic solvents,with or without the addition of a surfactant and other pharmaceuticallyacceptable adjuvants.

The compounds can also be administered in the form of a depot injectionor implant preparation, which may be formulated in such a manner as topermit a sustained release of the active ingredient.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, troches, powders,solutions, suspensions or emulsions.

For topical application the compounds can be administered in the form ofan unguent, cream, ointment, lotion or a patch.

The invention consequently also refers to a method of treatment of acancer in a mammal, comprising the steps of administrating apharmaceutical composition, containing a compound having the formula Iin combination with a physiologically acceptable carrier, by constantinfusion to a patient suffering from a tumor, controlling the plasmalevel of the compound, and adjusting the rate of infusion to keep theplasma level at a concentration of 0.05-5.0 μM, for a period of timebeing sufficient for the tumor to be retarded or to disappear.

EXPERIMENTAL Materials

Chemicals

Cell culture reagents, that is media, fetal calf serum and antibiotics,were purchased from Gibco, Sweden. All other chemicals unless statedotherwise were from Sigma (St. Louis. MO, USA). A mouse monoclonalantibody against phosphotyrosine (PY99) and a polyclonal antibodyagainst α-subunit of IGF-1R(N20) and α-subunit of the insulin receptor,and a polyclonal antibody against the platelet-derived growth factorreceptor were obtained from Santa Cruz Biotechnology Inc (Santa Cruz,Calif., USA). A monoclonal antibody against the α-subunit of IGF-1R(αIR-3) and a monoclonal antibody to fibroblast growth factor receptorwere purchased from Oncogene Science (Manhasset, N.Y., USA). The murinemonoclonal antibody against the epidermal growth factor receptor waspurchased from Life Science and the Anti-1RS-1 agarose conjugateantibody was obtained from UBI (Lake Placid, N.Y., USA).

(³H) Thymidine and (³H) leucine were from Amersham Int. (UK) and themonoclonal antibody against α-smooth muscle actin was from Sigma ImmunoChemicals (La Jolla, Calif., USA). Recombinant IGF-1 was a gift fromPharmacia Upjohn (Stockholm, Sweden). Deoxypodophyllotoxin andpodophyllotoxin (99.97% purity), and α-apopicropodophyllin,β-apopicropodophyllin, were kind gifts from Analytecon SA, Pre Jorat,Switzerland and so waspodophyllotoxin-4,6-0-benzylidene-β-D-glucopyranoside from Conpharm AB,Uppsala, Sweden. Etoposide, was from Sigma.

Cell Cultures

The human melanoma cell lines SK-MEL-2, SK-MEL-5 and SK-MEL28, theEwing's sarcoma cell lines RD-ES and ES-1, the hepatoma cell line HepG2,the prostatic carcinoma cell line PC-3, and the breast cancer cell lineMCF-7 were from the American Tissue Culture Collection, USA. Themalignant melanoma cell lines BE, DWB and FM 55 were obtained fromProfessor R Kiessling, CCK, Karolinska Hospital, Stockholm, Sweden. TheR—and P6 cell lines were gifts from Professor R. Baserga, ThomasJefferson University, Philadelphia, Pa., USA. R-cells are IGF-1Rnegative, whereas P6 cells overexpress the IGF-1R.

Keratinocytes (HaCaT cells) were provided by and tested in collaborationwith Professor Mona Backdahl, Department of Dermatology, KarolinskaHospital, Stockholm, Sweden. The HaCaT cell line is a spontaneouslyimmortalized human keratinocyte cell line (Boukamp P, et al., J CellBiol 106: 761-771, 1988), which is frequently used as an experimentalmodel for psoriasis (Wraight, C. J., et al. Nat Biotechnol 18: 521-526,2000.) HaCaT cells were cultured in Dulbecco's Modified Eagle's Mediumcontaining 10% fetal bovine serum, glutamine, benzylpenicillin andstreptomycin.

Human vascular smooth muscle cells (VSMC) were isolated and culturedfrom surgical specimen of human renal artery essentially as describedpreviously (Ross R., J Cell Biol 50: 172-186, 1971). Briefly, VSMC wereallowed to migrate from the primary explants and were subsequentlypassaged at confluence. Cells were maintained in F12 medium containing15% fetal bovine serum, 0.05 mg/ml ascorbic acid, 2 μg/ml fungizone and200 IU/ml of penicillin. Cultured cells were a uniform population ofhuman smooth muscle cells identified both by their morphology and byimmunostaining for smooth muscle specific alfa-actin, which recognizes aunique epitope. Culturing was performed at a temperature of 37° C.,humidity of 85% and at a CO₂ concentration of 5% in air. The medium waschanged twice a week and the cells were harvested at passage 2 to 8using a solution of trypsin (0.25%) and EDTA (0.02%).

The human chronic myeoloid leukemia K562/S and K562/Vcr30 lines and theacute myeloid leukemia cell lines HL60/0 and HL60/Nov were obtained fromATCC. The K562/S and K562/Vcr30 are wild type (non-resistent) cells,whereas K562/Vcr30 and HL60/Nov are cytostatic-resistant sublines. Allleukemia cell lines were cultured in RPMI 1640 medium supplemented with10% fetal bovine serum and with 2 mM L-glutamine, benzylpenicillin (100U/ml) and streptomycin (100 μg/ml). The cells were grown in tissueculture flasks maintained at 95% air/5% CO₂ atmosphere at 37° C. in ahumidified incubator. For the experiments cells were cultured in 60-mmplastic dishes or 96-well plastic plates.

All other cell lines were cultured in Minimal Essential Mediumcontaining 10% fetal bovine serum, glutamine, 1% benzylpenicillin andstreptomycin. The cells were grown in monolayers in tissue cultureflasks maintained at 95% air/5% CO₂ atmosphere at 37° C. in a humidifiedincubator. For the experiments cells were cultured in either 35-mm or60-mm plastic dishes or 96-well plastic plates. The experiments wereinitiated under subconfluent growth conditions.

Methods In Vitro Tyrosine Kinase Assays

IGF-1R-catalyzed substrate phosphorylation of polyTyrGlu (pTG) wasperformed essentially as previously described [Parrizas M., et al.,ibid., and Blum G., et al., ibid.]. Immunoprecipitated IR from HepG2,IGF-1R from P6 cell extract and immunodepleted supernatant to assaynon-IGF-1R tyrosine kinases. The phosphorylated polymer substrate wasprobed with a purified phosphotyrosine specific monoclonal antibodyconjugated to horseradish peroxidase (HARP). Color was developed withHRP chromogenic substrate o-phenylenediamine dihydrochloride (OPD). Thecolor was quantitated by spectrophotometry (ELISA reader) and reflectsthe relative amount of tyrosine kinase. The precipitate wasimmunoblotted with antibodies to IGF-1R and IR to verify the presence ofthe receptor. Serial dilutions were used to assay the optimal conditionswith respect to the amount of IGF-1R and IR. The signal was linear for30 minutes and was a function of IGF-1R concentration up to 75 ng/well.Briefly, 96 well plates (Immunolon, Nunc) were coated overnight at 4° C.with a mouse monoclonal antibody (LabVision) against the beta-subunit ofIGF-1R at a concentration of 1 μg/ml. The plates were blocked with BSAin PBS (ELISA blocking buffer, Pierce), and 80 μg/ml of total proteinlysate from the P6 cell line was added. The plates were incubated for 1h, and washed with PBS Tween. The investigated compounds were added inPBS at room temperature for 30 minutes, prior to kinase activation withIGF-1. Kinase assay was performed using the Sigma kit for in vitrophosphorylation following the manufacturer instructions. Afterspectrophotometry the IC₅₀ values of inhibitors were determined usingthe Regression function of Statistica program.

IGF-1R tyrosine autophosphorylation was analysed by a sandwich ELISAassay. Briefly, 96-well plates (Immunolon, Nunc) were coated overnightat 4° C. with 1 μg/well of the monoclonal antibody Ab-5 (LabVision) tothe IGF-1R beta subunit. The plates were blocked with 1% BSA in PBSTween for 1 h, then 80 g/well of total protein lysate from the P6 cellline was added. As a negative control was used total protein lysate fromthe R-cell line. The investigated compounds were added in tyrosinekinase buffer without ATP at room temperature for 30 min, prior tokinase activation with ATP. Kinase assay was performed using the Sigmakit. After spectrophotometry the IC50 values of inhibitors weredetermined using the Regression function of Statistica program.

Assay of Cell Growth and Survival

Cell proliferation kit II (Roche Inc.) is based on colorimetric changeof the yellow tetrazolium salt XTT in orange formazan dye by therespiratory chain of viable cells (Roehm, NR, et al., J Immunol Methods142: 257-265, 1991). Cells seeded at a concentration of 5000/well in 100gl medium in a 96-well plate were treated with different drugs in thegiven concentration. After 24 or 48 h the cells were incubated,according to the manufacturer's protocol, with XTT labelling mixture.After 4 h the formazan dye is quantified using a scanning multiwellspectrophotometer with a 495 nm filter. The absorbance is directlycorrelated with the number of viable cells. The standard absorbancecurve was drawn by means of untreated cells seeded at a concentration offrom 1000 to 10 000 cells/well with an increasing rate of 1000cells/well. All standards and experiments were performed in triplicates.

Assay of Tyrosine Phosphorylation of Receptors in Intact Cells

Cells were cultured to subconfluency in 6-cm plates, and then freshmedium containing 10% FBS and the desired compounds were added for 1 h.The cells were then lyzed and subjected to immunoprecipitation usingspecific antibodies. Immunoprecipitates were resolved by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferredto nitro-cellulose membranes and incubated with anti-phosphotyrosineantibody. Antibodies to actin (in cell extract) or IGF-1R beta subunitwere used as loading controls. After detection the films were scannedfor quantification.

Immunoprecipitation and Determination of Protein Content

The isolated cells were then lyzed in 10 ml ice-cold PBSTDS containingprotease inhibitors (Carlberg, M., et al., J Biol Chem 271: 17453-17462,1996). 50 μl protein A or G agarose was added in 1 ml sample andincubated for 15 min at 4° C. on an orbital shaker. After centrifugationfor 10 min at 10,000 r/min at 4° C. the supernatant was saved. Theprotein content was determined by a dye-binding assay with a reagentpurchased from Bio-Rad. Bovine serum albumin was used as a standard. 15μl Protein G Plus agarose and 5 μl anti-IGF-1R were added. After a 3 hincubation at 4° C. on an orbital shaker the precipitate was collectedby pulse centrifugation in a micro centrifuge at 14,000×g for 10 s. Thesupernatant was discarded and the pellet was washed 3 times with PBSTDS.

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Protein samples were solved in a 2×-sample buffer containing Laemmlibuffer and 0.5% methanol and boiled for 5 min at 96° C. Samples wereseparated by SDS-PAGE with a 4% stacking gel and 7.5% separation gel.Molecular weight markers (Bio Rad, Sweden) were run simultaneously inall experiments.

Western Blotting

Following SDS-PAGE the proteins were transferred overnight tonitro-cellulose membranes (Hybond, Amersham, UK) and then blocked for 1h at room temperature in a solution of 4 g skimmed milk powder and 0.02%Tween 20 in PBS, pH 7.5. Incubations with the primary antibodies wereperformed for 1 h at room temperature, followed by 3 washes with PBSwith Tween and incubation with the second antibody for 1 h roomtemperature. After another 3 washes the membranes were incubated withStreptavidin-labelled horseradish peroxidase for 30 min and thendetected using Amersham ECL system (Amersham, UK). The films werescanned by Fluor-S (BioRad).

Experiment 1 Effect of Podophyllotoxin Derivatives on Phosphorylation ofIGF-1R in Cultured Melanoma Cells

FM55 melanoma cells were seeded in 6-cm dishes, at a concentration of10,000 cells/cm² in Minimal Essential Medium supplemented with 10 ofetal calf serum (FCS). When the cells reached a density of 65,000cells/cm² in the dishes, they were treated for 1 h with 0.05 μMpodophyllotoxin, deoxypodophyllotoxin, picropodophyllin,deoxypicropodophyllin,4′-demethyl-7-(4,6-O-ethylidene-O-D-glucopyranosyl) epipodophyllotoxin(etoposide) and podophyllotoxin-4,6-O-benzylidene-O-D-glucopyranoside(pf-4, 6-0). Etoposide and pf-4, 6-0 were also administered at 15 μM.The cells were then harvested for assay and quantification of IGF-1Rphosphorylation as described in Experiment 1. The values shown in Table1 represent means of 3 experiments.

TABLE 1 Level of IGF-1R phosphorylation in intact cells (% OD)Podophyllotoxin 5 Deoxypodophyllotoxin 2 Picropodophyllin 8Deoxypicropodophyllin 5 Etoposide (0.05 μM) 102 Etoposide (15 μM) 105Pf-4,6-O (0.05 μM) 100 Pf-4,6-O (15 μM) 102

The results show that podophyllotoxin, deoxypodophyllotoxin,picropodophyllin and deoxypicropodophyllin are all potent inhibitors ofIGF-1R phosphorylation, while etoposide and Pf-4, 6-O are not.

Experiment 2 Dose-Response Effect of Picropodophyllin on Phosphorylationof IGF-1R in a Cell-Free System

All these data on intact cells showed that picropodophyllin andpodophyllotoxin prevented phosphorylation of IGF-1R, but did not revealif this was a direct or an indirect effect on the tyrosine kinase.Therefore we isolated the receptor and determined the effects ofpicropodophyllin on IGF-1R catalyzed substrate tyrosine phosphorylationand IGF-1R autophosphorylation in-vitro. Picropodophyllin efficientlydecreased the phosphorylation of the pTG substrate (IC₅₀ value 0.006 μM,see FIG. 3). In contrast, it failed to interfere with substratephosphorylation of EGFR and IR tyrosine kinases, as well as that ofother ‘non-IGF-1R kinases’ (FIG. 3), which were obtained byimmunodepletion of IGF-1R. Podophyllotoxin produced similar results aspicropodophyllin.

In the next set of cell-free experiments we demonstrated that PPPefficiently inhibited the autophosphorylation of IGF-1R (for details seeMethods), with an IC₅₀ value of around 0.001 μM (see FIG. 4). A similarresponse was obtained by PPT (data not shown). To investigate whetherPPP interferes with the tyrosine autophosphorylation at the ATP level orat the substrate level (i.e., the tyrosine kinase domain of the IGF-1Rβ-subunit), various concentrations of ATP (19-300 μM) were added to thereaction buffer during the assay. As shown, this did not alter the IC₅₀value of PPP, which remained at 0.001-0.002 μM (FIG. 4). These resultsimply that the inhibitors of IGF-1R autophosphorylation do not act byinterfering with ATP, but rather inhibit substrate phosphorylation bythe IGF-1R tyrosine kinase.

Experiment 3 Specificity of Picropodophyllin and Podophyllotoxin onVarious Receptor Tyrosine Kinases in Cultured Cells

FM55 melanoma cells were cultured in the same way as described inExperiment 1. When reaching a density of 65,000 cells/cm² in the dishes,they were treated for 1 h with 0 (control) and 0.05 μM ofpicropodophyllin and podophyllotoxin, respectively. The cells were thenisolated and subjected to immunoprecipitation of the IGF-1R, fibroblastgrowth factor receptor (FGFR), platelet-derived growth factor receptor(PDGFR), epidermal growth factor receptor (EGFR), insulin receptor (IR)and insulin substrate-1 (IRS-1) using antibodies to respectivemolecules. IRS-1 is a substrate of IGF-1R, and therefore itsphosphorylation is dependent on phosphorylated IGF-1R.Gelelectrophoresis, Western blotting and quantification of the differentsignals were performed as described above.

TABLE 2 Level of IGF-1R phosporylation in intact cells (% OD)Picropodophyllin (PPP) and Podophyllotoxin (PPT) 0.05 μM Substrate PPTPPP IGF-1R 7 20 FGFR 105 100 PDGFR 100 104 EGFR 107 106 IR 101 100 IRS-110 17

This demonstrates that picropodophyllin and podophyllotoxin are specificfor IGF-1R.

Experiment 4 Effects of Podophyllotoxin and Picropodophyllin on IGF-1RPhosphorylation of Various Malignant Cell Types

12 cell lines of different origins were seeded in 6-cm dishes, at aconcentration of 10,000 cells/cm² in Minimal Essential Mediumsupplemented with fetal calf serum (FCS). When the cells had reached aconcentration of 65,000 cells/cm², they were treated with 0, 0.01,0.025, 0.05, 0.1 or 1.0 uM doses of podophyllotoxin and picropodophyllinfor 1 h. The cells were then harvested for assay and quantification ofIGF-1R phosphorylation as described above. The EC50 value, the EfficientConcentration 50%, i.e. the concentration needed to reduce thephosphorylation with 50%, for each inhibitor and cell line, is thencalculated. The values are shown below in Table 3. The results are basedon two different experiments.

TABLE 3 EC50 for IGF-1R phosphorylation Cell line Origin PodophyllotoxinPicropodophyllin SK-MEL-2 melanoma 0.04 Nd SK-MEL-5 melanoma 0.03 0.04SK-MEL-28 melanoma 0.03 0.04 BE melanoma 0.04 nd FM55 melanoma 0.04 0.06DWB melanoma 0.03 nd MCF-7 Breast cancer 0.05 0.04 PC-3 prostate cancer0.06 0.09 RD-ES Ewing's sarcoma 0.03 0.05 HepG2 hepatoblastoma —* —* R-IGF-1R negative —* —* fibroblasts P6 fibroblasts with 0.02 0.03overexpressed IGF-1R *No IGF-1R activity in controls. nd, not determined

This shows that podophyllotoxin and picropodophyllin inhibits IGFIRphosphorylation in various cancer cells.

Experiment 5 Effects of Podophyllotoxin and Picropodophyllin onViability of a Large Number of Malignant Cell Types

12 different types of cell lines were seeded in 96-well plates (mediumvolume in a well was 100 μl), at a concentration of 10,000 cells/cm² inMinimal Essential Medium supplemented with fetal calf serum. When thecells had reached a concentration of 65,000 cells/cm², they were treatedwith different doses of podophyllotoxin and picropodophyllin for 48 h.Cell viability was then assayed (see above). EC50 values for eachinhibitor and cell line, calculated as the concentration, resulting in a50% decrease in cell survival, are shown below in Table 4. Thedose-response curves for the melanoma cell line FM55 and the Ewing'ssarcoma cell line

RD-ES treated with picropodophyllin are depicted in FIG. 5A. Theviability of both tumor cell lines is decreased by the picropodophyllinconcentration. FIG. 5B demonstrates the dose-response curves for amurine fibroblast cell line, either devoid of (R—) or overexpressing thehuman IGF-1R (P6). Whereas the viability of P6 cells drops withpicropodophyllin dose, the R-cells are not responsive. This implies thatpicropodophyllin is selective for the IGF-1R. All results presented inTable 4 and FIG. 5 are based on 4 different experiments.

TABLE 4 EC50 (μM) for cell viability Cell line Origin PodophyllotoxinPicropodophyllin SK-MEL-2 melanoma 0.05 Nd SK-MEL-5 melanoma 0.02 0.05SK-MEL-28 melanoma 0.02 0.03 BE melanoma 0.05 nd FM55 melanoma 0.04 0.07DWB melanoma 0.04 nd MCF-7 breast cancer 0.07 0.09 PC-3 prostate cancer0.06 0.09 RD-ES Ewing's sarcoma 0.10 0.10 HepG2 hepatoblastoma >15 >15R- IGF-1R negative >15 >15 fibroblasts P6 fibroblasts with 0.03 0.02overexpressed IGF- 1R * nd, not determined

Podophyllotoxin and picropodophyllin are very potent inhibitors of tumorcell viability.

Experiment 6 Inhibition of Malignant Cell Growth In Vivo

Four to five-week old pathogen-free nude mice (nu/nu) were used andhoused within plastic isolators in a sterile facility. The Ewing'ssarcoma cell line ES-1 and the melanoma cell line BE (both proved toexpress IGF-1R) were injected subcutaneously at 10⁷ cells/mice in a 0.2ml volume of sterile saline solution. Experimental treatments withpodophyllotoxin, deoxypodophyllotoxin or picropodophyllin were performedby daily intraperitoneal injections of the compound in 100 μl volume ofsolvent, composed of a mixture of DMSO and physiological saline (8:2).Control mice were treated with the solvent. 3-6 animals were treated ineach group. Animals were monitored three times a week for signs ofdisease and tumor growth. Tumor mass was estimated using the formula(d²×D)/2, where d and D represent the small and large diameters of thetumor, respectively. The mice were carefully observed for presence ofside effects and were sacrificed at the end of the experiments forhistological analysis of the lesions. All experiments were performedaccording to the ethical guidelines for laboratory animal use, providedby institutional ethical committee.

The first set of experiments was carried out to investigate possiblelocal and systemic toxic effects of podophyllotoxin,deoxypodophyllotoxin and picropodophyllin on nude mice. In the firstexperiment osmotic pumps loaded with either drug-free solvent,podophyllotoxin (0.25 mg), deoxypodophyllotoxin (0.25 mg) orpicropodophyllin (0.25 mg) were implanted subcutaneously in the flankregion. The drugs are delivered subcutaneously with a constant flow of0.6 μl/h over 7 days. The volume of the pumps was 100 μl. After 7 daysthe mice were killed and the skin and subcutaneous tissue adjacent tothe outlet of the pumps were analyzed and scored for tissue reactions byan experienced pathologist. There were 3 mice in each group. Treatmentwith the solvent (control) did not give rise to any damages. Incontrast, podophyllotoxin caused severe tissue reactions with necrosis,bleeding and inflammation (Table 5). Mice treated withdeoxypodophyllotoxin got light damages, while thepicropodophyllin-treated animals exhibited no visible reactions (Table5).

In the next experiment we analyzed systemic effects of the drugs byinjecting 100 μl intraperitoneally of each compound daily over 5 days.Two doses were injected (7 or 28 mg/kg/d) and 3-6 mice were used foreach drug and dose. The mice were checked carefully daily for signs ofdiscomfort, diseases and weight loss. It was first confirmed that themice tolerated the drug-free solvent well. However, mice treated withthe low as well as with the high dose of podophyllotoxin became sick andwithin 2 days 67% and 100%, respectively, of the animals were dead(Table 6). Low-dose deoxypodophyllotoxin-treated mice exhibited serioussigns of disease after 3 days. At the high dose the experiment wasstopped after 2 days because of serious disease or death. In contrast,mice treated with either dose of picropodophyllin survived the whole5-day experiments and did not exhibit any evidence of disease (Table 6).

Because of the toxicity of podophyllotoxin and deoxypodophyllotoxin, weonly used picropodophyllin to analyze effects on tumor xenografts. Forthis purpose ES-1 (Ewing's sarcoma cells) and BE (melanoma cells)xenografts were established in nude mice. When the ES-1 and BE tumorsstarted growing subcutaneously and were measurable, mice were treateddaily with intraperitoneal injections of picropodophyllin (28 mg/kg,which was required for giving an average concentration above 0.05 μM ofpicropodophyllin in the blood plasma) or 80% DMSO in saline as a carrierfor 4-6 days, followed by 4-6 days without treatment. The mice were thenkilled and the tumors analyzed by an experienced pathologist.Picropodophyllin significantly inhibited growth of both types of tumorsand caused regression (see FIGS. 6A and 6B), and the histologicalanalysis of the tumor specimens revealed massive necrosis.

The results show that picropodophyllin, in contrast to podophyllotoxinand deoxypodophyllotoxin, is well tolerated by the animals, and causestumor regression.

TABLE 5 Local toxicity Effects on skin and Compound subcutaneous tissue*Solvent 0 Podophyllotoxin (7 mg/kg/d) +++ Deoxypodophyllotoxin (7mg/kg/d) + Picropodophyllin (7 mg/kg/d) 0 0, no tissue reaction; *,light tissue reaction with hyperemia and mild inflammation; **, moderatetissue reaction with strong inflammation; ***, strong tissue reactionwith necrosis and bleedings.

TABLE 6 Systemic toxicity Surviving mice/ Investigated mice Compound anddose in mg/kg/d 1 d 2 d 3 d 4 d 5 d Solvent 6/6 6/6 6/6 6/6 6/6Podophyllotoxin, 7 mg 3/3 1/3 0/3 — — Podophyllotoxin, 28 mg 2/6 0/6 — —— Deoxypodophyllotoxin, 7 mg 3/3 3/3 3/3 3/3 3/3 Deoxypodophyllotoxin,28 mg 6/6 4/6 0/6 — — Picropodophyllin, 7 mg 3/3 3/3 3/3 3/3 3/3Picropodophyllin, 28 mg 6/6 6/6 6/6 6/6 6/6

Experiment 7 Effects of Picropodophyllin and Derivatives on Survival ofHuman Leukemia Cells

The leukemia cell lines (K562/S, K562/Vcr30, HL60/0 and HL60/Nov) allexpress the IGF-1R, as assayed by Western blotting analysis, asdescribed in Methods and Experiment 1 and 2.

Cells of said cell lines were seeded in 96-well plates (25 000cells/well, medium volume per well was 100 μl), in RPMI40 mediumsupplemented with fetal calf serum. After 24 h picropodophyllin and thederivatives alpha-apopicropodophyllin and beta-apopicropodophyllin wereadded at different concentrations and the cells were incubated for 72 h.Cell viability was then assayed (see above). EC50 values for eachcompound and cell line are shown below (Table 7). The results are basedon 3 different experiments.

The results, presented in Table 7, show that 0.11-0.32 μMpicropodophyllin is needed to cause 50% cell death in 3 of the 4 celllines, whereas more than 0.5 μM is required for thevincristine-resistant cell line K562/Vcr30. In contrast, the IC₅₀ valuesfor alpha- and beta-apopicropodophyllin were as low as 0.010.05 μM.

TABLE 7 IC₅₀ (μM) for cell viability K562/S K562/Vcr30 HL60 HL60/NovPicropodophyllin 0.32 >0.50 0.17 0.11 Alpha-apopicro 0.04 0.05 0.02 0.02Beta-apopicro 0.01 0.01 0.01 0.01

It can be concluded that the picropodophyllin derivatives alpha- andbeta-apopicropodophyllin are highly potent inhibitors of leukemia cellgrowth and survival.

Experiment 8 Interactive Effects of Picropodophyllin on Malignant CellsTreated with Cytostatica

The leukemia cell lines K562/S, K562/Nov, HL60/0 and HL60/Nov werecultured in 96-well plates as described in Experiment 7. After 24 h, thecells were treated for 72 h with different concentrations of theanti-cancer agent vincristine, with and without co-incubation with 0.05μM picropodophyllin. It was proven that picropodophyllin at thisconcentration did not cause any detectable cell death in the tumorcells. Cell viability was then assayed. IC₅₀ values for each inhibitorand cell line are shown below (Table 8). The results are based on 3different experiments.

As shown, co-incubation with picropodophyllin decreased IC₅₀ values forcell viability in all 4 cell lines.

TABLE 8 IC₅₀ (nM) for cell viability Picropodophyllin (0.05 μM) + Cellline Vincristine (IC₅₀, nM) Vincristine (IC₅₀, nM) K562/S 1.6 0.6K562/Vcr30 5.0 0.3 HL60/0 0.8 0.4 HL60/Nov 1.2 0.8

The results show that picropodophyllin sensitises malignant cells forconventional cytostatic agents.

Experiment 9 Effects of Picropodophyllin and Podophyllotoxin on IGF-1RPhosphorylation and Cell Survival of a Human Psoriasis Model Cell Line

HaCaT cells, which are immortalized human keratinocytes and represent amodel cell line for psoriasis, were seeded in 6-cm dishes or in 96-wellplates (medium volume in a well was 100 μl), at a concentration of 7,000cells/cm² in Dulbecco's Modified Eagle's Medium containing 10% fetalbovine serum. When the cells reached a concentration of 50,000cells/cm², they were incubated with podophyllotoxin or picropodophyllinto a final concentration of 0 or 0.05 μM in the culture medium.Treatment with 0 μM represents untreated controls. After 1 h incubationthe cells in the 6-cm dishes were harvested for assay and quantificationof IGF-1R phosphorylation as described in Experiment 1. After 48 hincubation the cells cultured in the 96-well plates were assayed forcell viability by means of the Cell proliferation kit II, as describedabove.

The results show that both podophyllotoxin and picropodophyllin areefficient inhibitors of IGF-1R phosphorylation and cell viability inHaCaT cells.

TABLE 9 Effects of 0.05 pM of podophyllotoxin and picropodophyllin onlevel of IGF-1R phosphorylation (1 h) and cell survival of HaCaT cells(48 h). IGF-lR Cell Compound phosphorylation viability Control 100 100Podophyllotoxin 45 30 Picropodophyllin 35 35

Experiment 10 Effects of Podophyllotoxin and Picropodophyllin on IGF-1RPhosphorylation and Cell Proliferation of Cultured Human Vascular SmoothMuscle Cells (VSMC) in a Model for Artheriosclerosis and Restenosis

IGF-1 is a growth promoter for arterial cells and a mediator ofcardiovascular diseases, such as coronary artheriosclerotic plaquedevelopment and restenosis after coronary angioplasty. A key role inthese events is played by an excessive growth of VSMC in the vesselwall, which is caused by IGF-1 (Bayes-Genis A, et al., ibid.). For theexperiment, the isolated and cultured VSMC are grown in 24-well plates(20.000-40.000 cells/well) and studies on effects of podophyllotoxin andpicropodophyllin on IGF-1R phosphorylation and growth and survival ofthe VSMC are performed essentially as described in Experiment 9. Inaddition, cell proliferation is assessed by measuring (³H) thymidineincorporation into DNA (DNA synthesis) and (³H) leucine incorporationinto proteins (protein synthesis). In the former case, the cells(20.000-40.000 cells/well) are grown in 24-well plates and incubated for24 h with the addition of 1 μCi/ml (³H) thymidine and of IGF-1 (nM-μMconcentrations; alone or present in fetal bovine serum) with and withoutpodophyllotoxin or picropodophyllin at different concentrations (0-1.0μM). The cells are then washed with F12-medium and DNA is precipitatedwith 5% ice cold trichloroacetic acid (TCA). DNA is solubilized in 0.1 MKOH and 500 μl of the solution in each well is added to scintillationliquid and the radioactivity determined in a liquid scintillationcounter. In the latter case, cells are incubated for 24 h as describedabove, but without (³H) thymidine. Instead (³H) leucine is added toreach a concentration of 1 μCi/ml, but only for the last 90 minutes ofthe incubation. The cells arc then rinsed with cold phosphate bufferedsaline (pH=7.4) and proteins are precipitated in ice-cold TCA. Theproteins are solubilized in a solution containing: 5% sodium dodecylsulphate, 20 mM Na₂CO₃ and 2 mM EDTA. Radioactivity is determined byliquid scintillation counting. The results on DNA and protein synthesisin VSMC are presented as % of control cells, i.e. those incubatedwithout podophyllotoxin or picropodophyllin.

CONCLUSION

It has been demonstrated that picropodophyllin, as well as derivativesthereof having a cis configuration in the lactone ring, are highlyspecific and potent inhibitors of the IGF-1R tyrosine kinase.

Picropodophyllin-induced inactivation of the insulin-like growthfactor-1 receptor caused extensive cell death in malignant cells,whereas cells devoid of insulin-like growth factor-1 receptors wereresistant. This new mechanism of picropodophyllin and derivativesthereof may be useful in therapy of cancer and other IGF-1R dependentdiseases.

1-16. (canceled)
 17. A composition, comprising: (a) a compound offormula I:

wherein each R₁ is, independently, OH or OCH₃, n is 0, 1 or 2, R₂ is Hor OH, R₃ and R₄, together, are an ether or a lactone, and wherein thecarbons in positions 9 and 9′ have a cis configuration and the 8-9 and8′-9′ bonds are beta bonds, and (b) an anti-cancer drug.
 18. Thecomposition of claim 17, wherein the compound of formula I ispicropodophyllin.
 19. The composition of claim 17, wherein the compoundof formula I is deoxypicropodophyllin.
 20. The composition of claim 17,wherein the anti-cancer drug is a cytostaticum.
 21. The composition ofclaim 20, wherein the cytostaticum is vincristin, taxol or etoposide.22. The composition of claim 17, wherein R₁ is OH.
 23. The compositionof claim 17, wherein R₁ is OCH₃.
 24. The composition of claim 17,wherein n is
 0. 25. The composition of claim 17, wherein n is
 1. 26. Thecomposition of claim 17, wherein n is
 2. 27. The composition of claim17, wherein R2 is H.
 28. The composition of claim 17, wherein R2 is OH.29. The composition of claim 17, wherein R3 and R4, together, are anether.
 30. The composition of claim 17, wherein R3 and R4, together, area lactone.
 31. The composition of claim 17, further comprising aphysiologically acceptable carrier.
 32. A kit, comprising (a) a firstcontainer comprising a compound of formula I

wherein each R₁ is, independently, OH or OCH₃, n is 0, 1 or 2, R₂ is Hor OH, R₃ and R₄, together, are an ether or a lactone, and wherein thecarbons in positions 9 and 9′ have a cis configuration and the 8-9 and8′-9′ bonds are beta bonds, and (b) a second container comprising ananti-cancer drug.
 33. The kit of claim 32, wherein the anti-cancer drugis a cytostaticum.
 34. The kit of claim 33, wherein the cytostaticum isvincristin, taxol or etoposide.
 35. A method of treating cancer,comprising administering an effective amount of the composition of claim17 to a subject in need thereof.
 36. A method of treating cancer,comprising administering to a subject in need thereof an effectiveamount of the compound of formula I and the anti-cancer drug from thekit of claim
 32. 37. A method of treating a tumor, comprisingadministering an effective amount of the composition of claim 17 to asubject in need thereof.
 38. A method of treating a tumor, comprisingadministering to a subject in need thereof an effective amount of thecompound of formula I and the anti-cancer drug from the kit of claim 32.